SUMMARY The dengue virus (DENV) is a mosquito-borne RNA virus that is responsible for over 390 million infections a year. While dengue fever is mostly self-limiting, severe dengue diseases are the leading causes of hospitalization and death in children living in limited resource areas. Currently, there are no antiviral treatments for dengue diseases and existing vaccines have questionable safety profiles. Due to its global prevalence and economic burden, the discovery of anti-dengue therapeutics is an urgent health priority. The DENV virus encodes nonstructural 3 (NS3), an enzymatic protein with helicase activity. Mutations in NS3's helicase active site prevents viral RNA synthesis initiation and render the virus replication-incompetent. Further, amino acid residues responsible for NS3's helicase activity are conserved across the DENV serotypes. Thus, NS3 is thought to be an attractive target for dengue drug discovery. However, hits identified from biochemical screens suffer from poor in vivo pharmacokinetics issues while hits identified from phenotypic screens are often non-specific. Therefore, a HTS assay that can specifically target NS3 helicase activity and be performed in cell-based formats would enable the identification of more pharmacoactive hits. This Phase I SBIR application aims to develop a genetically encodable fluorescent sensor that can report small molecule modulations of helicase activities and a proof-of-concept cell-based assay that can identify small molecules that inhibit NS3 helicase activity in vivo. We believe the eventual cell-based HTS platform would be a highly valuable tool in dengue drug discovery.